The primary aim of our research plan is directed towards an understanding of cell wall biosynthesis on the molecular level. This research will provide insights into the processing of peptidoglycan (PG), the mechanism of penicillin action, dynamics of the assembly processes, levels of lipid bilayer organization, and the biosynthesis and function of lipoteichoic acid. Five specific aims will be investigated. (1) It is our hypothesis that the cross-linking of PG in Gaffkya homari occurs by a pathway that requires a penicillin-sensitive N- acetylmuramyl-L-alanine amidase and a penicillin-insensitive transpeptidase. To support this hypothesis, it is our aim to isolate the amidase from this organism and relate this enzyme to PBP-6, the primary in vivo target of penicillin. (2) Penicillin also causes the enhanced secretion of water-soluble PG, lipids, LTA and proteins. It is our aim to establish the mechanisms of the penicillin-induced release of these compounds. (3) The intramembranal mechanism of peptidoglycan formation is not well understood. It is our goal to use probe-labeled lipid intermediates to study the dynamics of the assembly process, the formation of lipid oligomers, and the membrane proteins in the assembly complex. (4) Lipid bilayer organization plays an important role in the assembly of PG. It is our aim to develop an understanding of the organization that is responsible for the association of membrane and wall, and the organization of the PG assembly complex. (5) The D-alanyl esters of lipoteichoic acid appear to play a vital function in cell division and separation. It is our aim to establish the molecular events required for the incorporation of D-alanine and the transacylation of the D-alanyl ester residues. Transacylation of these esters on the poly(glycerophosphate) moiety may represent a unique way of transmitting a signal from one location in the membrane to another. The effects of antibiotics, e.g. penicillin, on well-characterized assembly systems is instrumental to our definition of their mechanisms of action. As new target sites are described n these systems, it is proposed that novel types of antibacterial agents may be designed. D-Alanyl-lipoteichoic acid has been implicated in a variety of bacterial adhesion processes involved in the infectious process. This property may be modulated in part by the D-alanyl ester residues.